Liposomal oligonucleotide compositions

ABSTRACT

A pharmaceutical composition comprising (A) an oligonucleotide 8 to 50 nucleotides in length, which is targeted to mRNA encoding human raf and is capable of inhibiting raf expression, entrapped in (B) sterically stabilized liposomes.

This invention relates to liposomal oligonucleotide compositions, theirpreparation and their use.

Alterations in cellular genes which directly or indirectly control cellgrowth and differentiation are considered to be the main cause ofcancer. There are some thirty families of genes, called oncogenes, whichare implicated in human tumor formation. Members of one such family, theraf gene family, are frequently found to be mutated in human tumors. Theraf family includes three highly conserved genes termed A-, B- and c-raf(also called raf-1). c-Raf, the best characterized member of the raffamily, is the cellular homologue of v-raf, the transforming gene of themurine sarcoma virus 3611. Raf genes encode protein kinases that arethought to play important regulatory roles in signal transductionprocesses that regulate cell proliferation. Mutation of raf genescausing a truncation or other modification that leads to the expressionof raf kinase without a functional negative regulatory domain at theamino-terminal end results in conversion to a form which is implicatedin transformation of mammalian cells in culture, and tumor formation. Araf gene having an absent or inactive regulatory domain is said to be"activated." Activated (truncated) raf has been detected in a variety ofhuman cancers including small-cell lung carcinoma, primary stomachcancer, renal cancer, breast cancer, laryngeal cancer, skin fibroblastsfrom members of a cancer-prone family (Li-Fraumeni syndrome), and in ahuman glioblastoma cell line. Abnormal expression of the normal(non-activated) c-raf protein is believed to play a role in abnormalcell proliferation since it has been reported that 60% of all lungcarcinoma cell lines express unusually high levels of normal c-raf mRNAand protein. Rapp et al., The Oncogene Handbook, E. P. Reddy, A. M.Skalka and T. Curran, eds., Elsevier Science Publishers, New York, 1988,pp. 213-253.

Oligonucleotides have been employed as therapeutic moieties in thetreatment of disease states in animals and man. For example, there havebeen identified antisense, triplex and other oligonucleotidecompositions which are capable of modulating expression of genesimplicated in viral, fungal and metabolic diseases. There remains a needfor compositions which can effectively inhibit abnormal raf geneexpression, i.e. inhibit expression of the activated raf product orinhibit unusually high level of expression of the normal raf product.

It has now been found that compositions which inhibit abnormal geneexpression and retain high anti-hyperproliferative activity afterprolonged circulation in the bloodstream can be prepared by formulationof oligonucleotides capable of inhibiting raf expression which aretargeted to mRNA encoding human raf within sterically stablisedliposomes. These compositions facilitate the reduction of accumulationof oligonucleotide in non-target organs and reduction of acute andchronic side effects during prolonged treatment.

Accordingly, the present invention provides a pharmaceutical compositioncomprising (A) an oligonucleotide 8 to 50 nucleotides in length, whichis targeted to mRNA encoding human raf and is capable of inhibiting rafexpression, entrapped in (B) sterically stabilised liposomes.

The relationship between an oligonucleotide and its complementarynucleic acid target to which it hybridises is commonly referred to as"antisense". Targetting an oligonucleotide to a chosen nucleic acidtarget may involve a multistep process. The process usually begins withidentifying a nucleic acid sequence whose function is to be modulated.This may be, as examples, a cellular gene (or mRNA made from the gene)whose expression is associated with a particular disease state, or aforeign nucleic acid from an infectious agent. In the present invention,the target is a nucleic acid encoding raf; in other words, the raf geneor mRNA expressed from the raf gene. The targeting process also includesdetermination of a site or sites within the nucleic acid sequence forthe oligonucleotide interaction to occur such that the desiredeffect--inhibition of abnormal raf gene expression-will result. Once thetarget site or sites have been identified, oligonucleotides are chosenwhich are sufficiently complementary to the target, i.e., hybridizesufficiently well and with sufficient specificity, to give the desiredinhibition.

Inhibition of abnormal raf gene expression can be measured in ways whichare routine in the art, for example by Northern blot assay of mRNAexpression or Western blot assay of protein expression. Effects on cellproliferation or tumor cell growth can also be measured, as describedhereinafter in the Examples. "Hybridization," in the context of thisinvention, means hydrogen bonding, also known as Watson-Crick basepairing, between complementary bases, usually on opposite nucleic acidstrands or two regions of a nucleic acid strand. Guanine and cytosineare examples of complementary bases which ar e known to form threehydrogen bonds between them. Adenine and thymine are examples ofcomplementary bases which form two hydrogen bonds between them."Specifically hybridizable" and "complementary" are terms which are usedto indicate a sufficient degree of complementarity such that stable andspecific binding occurs between the DNA or RNA target and theoligonucleotide. It is understood that an oligonucleotide need not be100% complementary to its target nucleic acid sequence to bespecifically hybridizable. An oligonucleotide is specificallyhybridizable when binding of the oligonucleotide to the targetinterferes with the normal function of the target molecule to cause aloss of utility, and there is a sufficient degree of complementarity toavoid non-specific binding of the oligonucleotide to non-targetsequences under conditions in which specific binding is desired, i.e.,under physiological conditions in the case of in vivo assays ortherapeutic treatment, or, in the case of in vitro assays, underconditions in which the assays are conducted.

In preferred embodiments of this invention, the oligonucleotide (A) istargeted to mRNA encoding c-raf or A-raf. In accordance with thisinvention, persons of ordinary skill in the art will understand thatmRNA includes not only the coding region which carries the informationto encode a protein using the three letter genetic code, but alsoassociated ribonucleotides which form a region known to such persons asthe 5'-untranslated region, the 3'-untranslated region, the 5' capregion, intron regions and intron/exon or splice junctionribonucleotides. Thus, oligonucleotides may be formulated in accordancewith this invention which are targeted wholly or in part to theseassociated ribonucleotides as well as to the coding ribonucleotides. Inpreferred embodiments, the oligonucleotide is targeted to a translationinitiation site (AUG codon) or sequences in the 5'- or 3'-untransiatedregion of th e human c-raf mRNA. Th e functions of messen ger RNA to beinterfered with include all vital functions such as translocation of theRNA to the site for protein translation, actual translation of proteinfrom the RNA, splicing or maturation of the RNA and possibly evenindependent catalytic activity which may be engaged in by the RNA. Theoverall effect of such interference with the RNA function is to causeinterference with raf protein expression. Oligonucleotides targeted tomRNA encoding human A-raf and, especially, human c-raf are presentlypreferred; however, compositions for modulating expression of otherforms of raf are also believed to have utility and are comprehended bythis invention.

In the context of this invention, the term "oligonucleotide" refers toan oligomer or polymer of nucleotide or nucleoside monomers consistingof naturally occurring bases, sugars and intersugar (backbone) linkages.The term "oligonucleotide" also includes oligomers comprisingnon-naturally occurring monomers, or portions thereof, which functionsimilarly. Such modified or substituted oligonucleotides are oftenpreferred over native forms because of properties such as, for example,enhanced cellular uptake and increased stability in the presence ofnucleases.

In some preferred oligonucleotides (A), at least one nucleotide ismodified at the 2' position of the sugar moiety. Certain preferredoligonucleotides (A) are chimeric oligonucleotides. "Chimericoligonucleotides" or "chimeras", in the context of this invention, areoligonucleotides which contain two or more chemically distinct regions,each made up of at least one nucleotide. These oligonucleotidestypically contain at least one region of modified nucleotides thatconfers one or more beneficial properties (such as, for example,increased nuclease resistance, increased uptake into cells, increasedbinding affinity for the RNA target) and a region that is a substratefor RNase H cleavage. In one preferred embodiment, a chimericoligonucleotide comprises at least one region modified to increasetarget binding affinity, and usually, a region that acts as a substratefor RNAse H. Affinity of an oligonucleotide for its target (in this casea nucleic acid encoding raf) is routinely determined by measuring the Tmof an oligonucleotide/target pair, which is the temperature at which theoligonucleotide and target dissociate; dissociation is detectedspectrophotometrically. The higher the Tm, the greater the affinity ofthe oligonucleotide for the target. In a more preferred embodiment, theregion of the oligonucleotide which is modified to increase raf mRNAbinding affinity comprises at least one nucleotide modified at the 2'position of the sugar, particularly a 2'-alkoxy, 2'-alkoxyalkoxy or2'-fluoro-modified nucleotide. Such modifications are routinelyincorporated into oligonucleotides and these oligonucleotides have beenshown to have a higher Tm (i.e., higher target binding affinity) than2'-deoxyoligonucleotides against a given target. The effect of suchincreased affinity is to greatly enhance antisense oligonucleotideinhibition of raf gene expression. RNAse H is a cellular endonucleasethat cleaves the RNA strand of RNA:DNA duplexes; activation of thisenzyme therefore results in cleavage of the RNA target, and thus cangreatly enhance the efficiency of antisense inhibition. Cleavage of theRNA target can be routinely demonstrated by gel electrophoresis. Inanother preferred embodiment, the chimeric oligonucleotide is alsomodified to enhance nuclease resistance. Cells contain a variety of exo-and endo-nucleases which can degrade nucleic acids. A number ofnucleotide and nucleoside modifications have been shown to make theoligonucleotide into which they are incorporated more resistant tonuclease digestion than the native oligodeoxynucleotide. Nucleaseresistance is routinely measured by incubating oligonucleotides withcellular extracts or isolated nuclease solutions and measuring theextent or isolated nuclease solutions and measuring the extent of intactoligonucleotide remaining over time, usually by gel electrophoresis.Oligonucleotides which have been modified to enhance their nucleaseresistance survive intact for a longer time than unmodifiedoligonucleotides. A variety of oligonucleotide modifications have beendemonstrated to enhance or confer nuclease resistance. Oligonucleotideswhich contain at least one phosphorothioate modification are presentlymore preferred. In some cases, oligonucleotide modifications whichenhance target binding affinity are also, independently, able to enhancenuclease resistance.

Specific examples of some preferred oligonucleotides may containphosphorothioate phosphotriester, methyl phosphonate, short chain alkylor cycloalkyl intersugar linkages or short chain heteroatomic orheterocyclic intersugar ("backbone") linkages. Most preferred arephosphorothioates and those with CH₂ --NH--O--CH₂, CH₂ --N(CH₃)--O--CH₂,CH₂ --O--N(CH₃)--CH₂, CH₂ --N(CH₃)--N(CH₃)--CH₂ and O--N(CH₃)--CH₂ --CH₂backbones (where phosphodiester is O--P--O--CH₂). Also preferred areoligonucleotides having morpholino backbone structures, for example asdescribed in U.S. Pat. No. 5,034,506. In other preferred embodiments,such as the protein-nucleic acid or peptide-nucleic acid (PNA) backbone,the phosphodiester backbone of the oligonucleotide may be replaced witha polyamide backbone, the bases being bound directly or indirectly tothe aza nitrogen atoms of the polyamide backbone, as described by P. E.Nielsen, M. Egholm, R. H. Berg, O. Buchardt, Science 1991, 254, 1497.Other preferred oligonucleotides may contain alkyl andhalogen-substituted sugar moieties comprising one of the following atthe 2' position: OH, SH, SCH₃, F, OCN, OCH₂ OCH₃, OCH₂ CH₂ OCH₃, OCH₂O(CH₂)_(n) CH₃, O(CH₂)_(n) NH₂ or O(CH₂)_(n) CH₃ where n is from 1 toabout 10; C₁ to C₁₀ lower alkyl, substituted lower alkyl, alkaryl oraralkyl; Cl; Br; CN; CF₃ ; OCF₃ ; O--, S--, or N-alkyl; O-, S-, orN-alkenyl; SOCH₃ ; SO₂ CH₃ ; ONO₂ ; NO₂ ; N₃ ; NH₂ ; heterocycloalkyl;heterocycloalkaryl; aminoalkylamino; polyalkylamino; substituted silyl;an RNA cleaving group; a cholesteryl group; a conjugate; a reportergroup; an intercalator; a group for improving the pharmacokineticproperties of an oligonucleotide; or a group for improving thepharmacodynamic properties of an oligonucleotide and other substituentshaving similar properties. Oligonucleotides may also have sugar mimeticssuch as cyclobutyls in place of the pentofuranosyl group. Otherpreferred embodiments may include at least one modified base form or"universal base" such as inosine.

In certain especially preferred embodiments of the invention, allnucleotides of the oligonucleotide (A) are 2'-deoxynucleotides and allbackbone linkages are phosphorothioate linkages.

In certain other especially preferred embodiments, the oligonucleotide(A) is a chimeric oligonucleotide having one or more regions with2'-deoxynucleotides and one or more regions with2'-alkoxyalkoxynucleotides, particularly 2'-methoxyethoxynucleotides,the one or more, 2'-deoxynucleotide regions preferably havingphosphorothioate backbone linkages and the one or more2'-alkoxyalkoxynucleotide regions preferably having phosphodiesterbackbone linkages. These chimeric oligonucleotides preferably comprise aregion of 2'-deoxynucleotides between two regions of2'-alkoxyalkoxynucleotides.

The oligonucleotides used as component (A) of the composition of theinvention may be conveniently and routinely made using well-knowntechniques such as solid phase synthesis. Equipment for such synthesisis available commercially from various sources including AppliedBiosystems. The use of such techniques to prepare oligonucleotides suchas the phosphorothioates and alkylated derivatives is well known. It isalso well known to use similar techniques and commercially availablemodified amidites and controlled-pore glass (CPG) products such asbiotin, fluorescein, acridine or psoralen-modified amidites and/or CPG(available from Glen Research, Sterling VA) to synthesize fluorescentlylabeled, biotinylated or other modified oligonucleotides such ascholesterol-modified oligonucleotides.

Specific especially preferred oligonucleotides, for which the nucleotidesequences have been published in WO 95/32987, include the following:

    ______________________________________                                        No.   Sequence (5' → 3')                                                                       Site    SEQ ID NO:                                    ______________________________________                                        ON1   GCTCCATTGATGCAGCTTAA                                                                            AUG     1                                             ON2          GATGCAGCTTAAACAATTCT                                                                          5'UTR                                                                                2                                         ON3          TCCCGCCTGTGACATGCATT                                                                          3'UTR                                                                                3                                         ON4          GTCTGGCGCTGCACCACTCT                                                                          3'UTR                                                                                4                                         ON5          CGCTCCTCCTCCCCGCGGCG                                                                          5'UTR                                                                                5                                         ON6          TCCTCCTCCCCGCGGCGGGT                                                                          5'UTR                                                                                6                                         ON7          CTCGCCCGCTCCTCCTCCCC                                                                          5'UTR                                                                                7                                         ON8          CTGGCTTCTCCTCCTCCCCT                                                                          3'UTR                                                                                8                                         ON9          CGGGAGGCGGTCACATTCGG                                                                          5'UTR                                                                                9                                         ON10        TCTGGCGCTGCACCACTCTC                                                                           3'UTR                                                                                10                                        ______________________________________                                    

ON1 to ON10 are oligodeoxynucleotides with phosphorothioate backbonesdesgined using the Genbank c-raf sequence HUMRAFR (Genbank listing x03484), synthesised and tested for inhibition of c-raf mRNA expressionin T24 bladder carcinoma cells using a Northern blot assay.

Other specific especially preferred oligonucleotides include:

    ______________________________________                                        No.   Sequence          Site    SEQ ID NO:                                    ______________________________________                                        ON11  CGGGAGGCGGTCACATTCGG                                                                            5'UTR   9                                             ON12          GATGCAGCTTAAACAATTCT                                                                       5'UTR                                                                                    2                                       ON13          GCTCCATTGATGCAGCTTAA                                                                        AUG        1                                      ON14          CGCTCCTCCTCCCCGCGGCG                                                                       5'UTR                                                                                    5                                       ON15          CGGGAGGCGGTCACATTCGG                                                                       5'UTR                                                                                   9                                        ______________________________________                                    

ON11, ON12 and ON13 are oligonucleotides synthesised withphosphorothioate backbones and uniformly substituted at the 2' positionof the sugar moiety by a methoxy group. ON14 is synthesized with aphosphodiester backbone and is uniformly substituted by a propoxy groupat the 2' position of the sugar moiety. ON15 is synthesized with aphosphorothioate backbone and is uniformly substituted by fluoro at the2' position of the sugar moiety.

Specifically especially preferred chimeric oligonucleotides include:

    ______________________________________                                        No.   Sequence         Target Site                                                                              SEQ ID NO:                                  ______________________________________                                        ON16  TCCTCCTCCCCGCGGCGGGT                                                                           5'UTR      6                                           ON17      CTCGCCCGCTCCTCCTCCCC                                                                        5'UTR            7                                    ON18      TTCTCGCCCGCTCCTCCTCC                                                                        5'UTR            11                                   ON19      TTCTCCTCCTCCCCTGGCAG                                                                        3'UTR            12                                   ON20      CTGGCTTCTCCTCCTCCCCT                                                                        3'UTR            8                                    ON21      CCTGCTGGCTTCTCCTCCTC                                                                        3'UTR            13                                   ON22      TCCCGCCTGTGACATGCATT                                                                        3'UTR            3                                    ON23      TCCCGCCTGTGACATGCATT                                                                        3'UTR            3                                    ON24      TCCCGCCTGTGACATGCATT                                                                        3'UTR            3                                    ON25      TCTGGCGCTGCACCACTCTC                                                                        3'UTR            10                                   ______________________________________                                    

ON16 to ON25 are chimeric oligonucleotides with uniform phosphorothiatebackbones, the nucleotides shown underlined being substituted by methoxyat the 2' position of the sugar moiety.

Other specific especially preferred chimeric oligonucleotides include:

    ______________________________________                                        No.   Sequence         Target Site                                                                              SEQ ID NO:                                  ______________________________________                                        ON26  TCCCGCCTGTGACATGCATT                                                                           3'UTR       3                                          ON27     TCCCGCCTGTGACATGCATT                                                                          3'UTR            3                                   ON28     TCTGGCGCTGCACCACTCTC                                                                          3'UTR           10                                   ______________________________________                                    

ON26, ON27 and ON28 are chimeric oligonucleotides with uniformphosphorothioate backbones, the nucleotides shown underlined beingsubstituted at the 2' position of the sugar moiety, in ON26 by propoxyand in ON27 and ON28 by fluoro.

Specific preferred chimeric oligonucleotides with 2' modifications andchimeric phosphorothiote/phosphodiester backbones include:

    ______________________________________                                        No.   Sequence         Target Site                                                                              SEQ ID NO:                                  ______________________________________                                        ON29  TCCCGCCTGTGACATGCATT                                                                           3'UTR       3                                          ON30    TCTGGCGCTGCACCACTCTC                                                                            3'UTR           10                                  ON31    TCCCGCCTGTGACATGCATT                                                                            3'UTR            3                                  ______________________________________                                    

ON29 and ON30 have regions, shown underlined, which have both 2'-propoxysubstituents and phosphodiester backbones. ON31 has regions, shownunderlined, which have both 2'-methoxyethoxy substituents andphosphodiester backbones.

It is believed that certain oligonucleotides targeted to portions of theA-raf mRNA and which inhibit A-raf expression will be useful forinterfering with cell hyperproliteration.

Specific phosphorthioate deoxyoligonucleotides of this kind, designedand synthesised using the Genbank A-raf sequence HUMARAFIR (Genbanklisting x 04790), for which the nucleotide sequences have been publishedin WO 95/32987, include the following:

    ______________________________________                                        No.   Sequence            Target Site                                                                              SEQ ID NO:                               ______________________________________                                        ON32  CCA TCC CGG ACA GTC ACC AC                                                                        Coding     15                                       ON33     ATG AGC TCC TCG CCA TCC AG                                                                     Coding                    16                        ON34     AAT GCT GGT GGA ACT TGT AG                                                                     Coding                    17                        ON35     CCG GTA CCC CAG GTT CTT CA                                                                     Coding                    18                        ON36     CTG GGC AGT CTG CCG GGC CA                                                                     Coding                    19                        ON37     CAC CTC AGC TGC CAT CCA CA                                                                     Coding                    20                        ON38     GAG ATT TTG CTG AGG TCC GG                                                                     Coding                    21                        ON39     GCA CTC CGC TCA ATC TTG GG                                                                     Coding                    22                        ON40     CTA AGG CAC AAG GCG GGC TG                                                                      Stop                      23                       ON41     ACG AAC ATT GAT TGG CTG GT                                                                      3'UTR                    24                        ON42     GTA TCC CCA AAG CCA AGA GG                                                                      3'UTR                    25                        ON43     GTC AAG ATG GGC TGA GGT GG                                                                      5'UTR                    14                        ______________________________________                                    

In compositions of the invention, the oligonucleotide (A) is entrappedin sterically stablised liposomes (B). Examples of sterically stabilisedliposomes are those in which part of the lipid is a glycolipid,particularly ganglioside GM, saturated phosphatidylinositol orgalactocerebroside sulphate ester, such as those described in WO88/04924; those in which part of the lipid is derivatised withhydrophilic polymer such as those described in WO 91/05545 or U.S. Pat.No. 5,225,212; and those comprising a vesicle-forming lipid and alipid-polymer conjugate having a hydrophobic moiety and a polar headgroup, such as those described in WO 94/20073.

In a preferred embodiment of the invention, the liposomes (B) compriseat least one underivatised vesicle-forming lipid and at least onevesicle-forming lipid derivatised with hydrophilic polymer which may be,for example, a polymer containing a hydroxy and/or carboxyl group suchas a polylactic acid, a polyglycolic acid or, preferably, a polyethyleneglycol. More preferably, the hydrophilic polymer is a polyethyleneglycolhaving a molecular weight of 1000 to 5000 daltons, such as 1500 to 2500daltons, especially 1800 to 2200 daltons. The hydrophilic polymer ispreferably derivatised with a polar head group of a phospholipid,especially a phospholipid having an amino head group, i.e. thederivatised lipid is preferably a phospholipid having an amino group,especially a phosphatidylethanolamine such as dilauroylphosphatidylethanolamine, dimyristoyl phosphatidylethanolamine, dioleoylphosphatidylethanolamine or, particularly, distearoylphosphatidylethanolamine.

Various methods of derivatising an amino-containing lipid with ahydroxyl- and/or carboxyl-containing hydrophilic polymer will beapparent to those skilled in the art. Several such methods are describedin WO 91/05545 and U.S. Pat. No. 5,225,212; the phospholipid having anamino group may be derivatised with the hydrophilic polymer by any ofthese methods. Preferably, the phospholipid having an amino group isderivatised with a hydroxyl-containing hydrophilic polymer such that thepolymer is attached to the phospholipid through a carbamate linkage;this may be achieved by reacting a hydroxyl group of the polymer (otherhydroxyl groups being capped, if necessary in view of their reactivity,for example by etherification) with diimidazole to give an activatedimidazole--terminated polymer which is then reacted with theamino-containing phospholipid to couple the phospholipid to thehydrophilic polymer through a carbamate group, as described in WO91/05545 or U.S. Pat. No. 5,225,212. In an especially preferredembodiment of the invention, the derivatised lipid is anamino-containing phospholipid, particularly a phosphatidylethanolamine,coupled through a carbamate group to a polyethyleneglycol capped at oneend by an alkoxy group, particularly a methoxy or ethoxy group. Such aderivatised lipid is available commercially.

The derivatised lipid is generally present in a minor molar amountrelative to the total Lipid content of the liposomes, preferably in anamount of 1 to 20 mole % of the total lipid content, although a loweramount, for example 0.1 mole %, may be appropriate when the derivatisedlipid has a high molecular weight. The major part of the lipid contentof the liposomes generally comprises one or more underivatisedvesicle-forming lipids such as are used in conventional liposomes. Suchlipids include, for example, lipids having two hydrocarbon chains,usually in acyl groups, and a polar head group, including phospholipids,for example phosphatidylcholines such as dilauroyl phosphatidylcholine,dimyristoyl phosphatidylcholine, dipalmitoyl phosphatidyicholine,distearoyl phosphatidylcholine, dioleoyl phosphatidylcholine,dilinoleoyl phosphatidylcholine, 1-palmitoyl-2-oleoylphosphatidylcholine, phosphatidylethanolamines such as those mentionedhereinbefore, and phosphatidic acids such as dimyristoyl phosphatidicacid and dipalmitoyl phosphatidic acid. Other conventionally used lipidsinclude sterols, particularly cholesterol, and glycolipids such as thosementioned hereinbefore. Preferably, the underivatised lipid comprises amixture of a phospholipid, especially a phosphatidylcholine, and asterol, especially cholesterol.

In the abovementioned preferred embodiment, the sterically stabilisedliposomes (B) preferably comprise 4-10 mol % of the derivatised lipid,40-80 mol % of the underivatised phospholipid and 20-50 mol % of thesterol. In especially preferred liposomes (B), the molar ratio ofderivatised lipid: underivatised phospholipid: sterol is 1:10:5.

In another preferred embodiment of the invention, the liposomes (B)comprise (i) a glycolipid together with (ii) a vesicle-formingphospholipid or sphingolipid or mixture thereof and, optionally, (iii) asterol and/or an acylglycerol lipid. The glycolipid is preferably anegatively charged glycolipid, especially ganglioside GM₁(monosialoganglioside) or hydrogenated phosphatidylinositol. Thevesicle-forming phospholipid may be one or more of the phospholipidshereinbefore mentioned, preferably a phosphatidylcholine, aphosphatidylethanolamine or a mixture thereof. Especially preferredphospholipids are distearoyl phosphatidylcholine and dioleoylphosphatidylethanolamine. The sphingolipid is preferably sphingomyelinand is preferably used together with a phospholipid. The sterol may be,for example, ergosterol or, preferably, cholesterol. The acylglycerollipid may be an ester of glycerol containing two fatty acid acyl groupseach having at least 12 carbon atoms, for example lauroyl, myristoyl,palmitoyl or oleoyl groups, and one acyl group of formula R¹ CO--, whereR¹ is a residue, containing up to 10 carbon atoms, of a monocarboxylicacid of formula R¹ COOH after removal of the --COOH group or,preferably, of formula --COR² COOH where R² is a residue, containing upto 10 carbon atoms, preferably 1 to 4 carbon atoms, of a dicarboxylicacid of formula HOOC--R² --COOH, especially succinic acid, after removalof both --COOH groups. An especially preferred acylglycerol is1,2-dipalmitoyl-sn-3-succinyl glycerol.

In this second preferred embodiment of the invention, the liposomespreferably comprise (i) a negatively charged glycolipid together with(ii) a vesicle-forming phospholipid and/or sphingolipid and (iii) asterol or acylglycerol lipid, especially (i) ganglioside GM₁ orhydrogenated phosphatidylinositol together with (ii) distearoylphosphatidylcholine or dioleoyl phosphatidylethanolamine or a mixturethereof with sphingomyelin and (iii) cholesterol or1,2-dipalmitoyl-sn-3-succinylglycerol.

The liposomes may comprise from 2 to 20 mol% of the glycolipid (i) and80 to 98 mol% of (ii) the phospholipid, sphingolipid or mixture thereof.In preferred embodiments, where the liposomes also comprise a sterol oracylglycerol, they may comprise 2 to 20 mol %, preferably 4 to 10 mol %,of the glycolipid, 40 to 80 mol %, preferably 60 to 80 mol %, of thephospholipid, sphingolipid or mixture thereof and 10 to 50 mol %,preferably 20 to 40 mol%, of the sterol or 5 to 40 mol %, preferably 10to 30 mol %, of the acylglycerol.

Specific especially preferred liposomes (B) are those describedhereinafter in the Examples.

The oligonucleotide-containing liposomes of the invention can beprepared using known methods for the preparation of drug-containingliposomes. For example, in one method, the lipid composition isdissolved in an organic solvent, such as an alcohol, ether,halohydrocarbon or mixture thereof, the solvent is removed from theresulting solution, for example by rotary evaparation or freeze drying,and the resulting lipid film is hydrated by dispersing in an aqueousmedium, such as phosphate-buffered saline or an aqueous solution of asugar, e.g. lactose, which medium also contains the oligonucleotide (A),to give an aqueous suspension of liposomes in the form of multilamellarvesicles (MLV's). The aqueous liposome suspension may be treated toreduce the liposome size, for example to give small unilamellar vesicles(SUV's), using known methods, for example by sonication or by extrusionthrough one or more membranes, e.g. polycarbonate membranes, having aselected pore size. Liposomes according to the invention preferably haveon average a particle size below 500 nm, more preferably 50 to 200 nm,especially 80 to 120 nm.

It is generally desirable to have as high a weight ratio ofoligonucleotide to lipid as possible consistent with liposome stability.The maximum for this weight ratio may vary depending on the nature andcomposition of the lipid component, but in general this maximum islikely to be about 1:20. Ratios between 1:40 and 1:400 can be used withgood results.

The invention includes a method of inhibiting the expression of humanraf which comprises contacting tissues or cells which express human rafwith a composition of the invention as hereinbefore defined. Theinvention also includes a method of treating mammalian cancer whichcomprises administering a composition of the invention as hereinbeforedefined to a mammal, particularly a human, in need of such treatment.

The composition of the invention may be administered by pulmonarydelivery or, preferably, parenterally, for example intravenously,subcutaneously, intraperitoneally or intramuscularly. The dosage dependsprincipally on the method of administration and on the severity andresponsiveness of the condition to be treated. Individual doses and theadministration regime can best be determined by individual judgement ofa particular case of illness. Diseases which may be treated with thecomposition include mammalian cancer, particularly human cancer such aslung cancer, stomach cancer, renal cancer, breast cancer, laryngealcancer, pancreatic cancer, colorectal cancer and malignant melanoma.

The invention is illustrated by the following Examples.

EXAMPLE 1

A derivatised lipid, prepared by coupling distearoylphosphatidylethanolamine to a methoxy-capped polyethylene glycol ofmolecular weight 2000 through a carbamate group (DSPE-MPEG 2000available from Genzyme), distearoyl phosphatidylcholine (available fromSigma Chemical) and cholesterol are dissolved, at a molar ratio of1:10:5, in chloroform. The solvent is removed by rotary evaporation toleave a lipid film. This film (250 mg) is hydrated with Hanks' balancedsalt solution (2 ml) buffered to pH 7.4 with 25 mM4-(2-hydroxyethyl)piperazine-1-ethane sulphonic acid (HEPES) andcontaining oligonucleotide ON3 as hereinbefore defined (1.2 mg). Theresulting MLV's are subjected to ten liquid nitrogen-water freeze-thawcycles and then sonicated (wavelength 6 μm) for 2 minutes to give smallunilamellar vesicles (SUVs) having an average diameter of 80 to 100 nm.The resulting liposomes are purified to remove unentrappedoligonucleotide by size exclusion chromatography using a Sephadex G-150column and a 25 mM sodium borate elution buffer.

Human lung adenocarcinoma A549 cells are implanted subcutaneously underthe dorsal outer skin of nude mice. A suspension of theoligonucleotide-containing liposomes in phosphate buffered saline isadministered by intravenous injection at a dosage of 0.60 mg/kg oncedaily beginning on day 10 after tumour cell inoculation. Tumour size ismeasured and tumour volume calculated on days 10, 14, 17, 21, 24 and 27following tumour cell inoculation. The above test is repeated with theoligonucleotide-containing liposomes being administered at a dosage of0.60 mg/kg a) every second day, b) every third day and c) once weekly,beginning on day 10 following tumour cell inoculation. The above testprocedure is repeated using a solution of oligonucleotide ON3, insteadof liposomes containing ON3, in phosphate buffered saline. The testresults are as follows:

    ______________________________________                                        Daily Administration                                                                      Tumour Volume (cm.sup.3)                                          Days After Inoculation                                                                    ON3            ON3 in Liposomes                                   ______________________________________                                        10          0.1130         0.1160                                             14          0.0830         0.0700                                             17          0.0850         0.0580                                             21          0.1210         0.0510                                             24          0.1340         0.0610                                             27          0.1560         0.0670                                             ______________________________________                                    

    ______________________________________                                        Administration Every Second Day                                                           Tumour Volume (cm.sup.3)                                          Days After Inoculation                                                                    ON3            ON3 in Liposomes                                   ______________________________________                                        10          0.1160         0.1270                                             14          0.1010         0.0810                                             17          0.1160         0.0810                                             21          0.1690         0.0820                                             24          0.2540         0.0920                                             27          0.3300         0.1090                                             ______________________________________                                    

    ______________________________________                                        Administration Every Third Day                                                            Tumour Volume (cm.sup.3)                                          Days After Inoculation                                                                    ON3            ON3 in Liposomes                                   ______________________________________                                        10          0.1230         0.1130                                             14          0.1150         0.0700                                             17          0.1750         0.0700                                             21          0.2260         0.0710                                             24          0.4290         0.0970                                             27          0.6650         0.1210                                             ______________________________________                                    

    ______________________________________                                        Weekly Administration                                                                     Tumour Volume (cm.sup.3)                                          Days After Inoculation                                                                    ON3            ON3 in Liposomes                                   ______________________________________                                        10          0.1300         0.1230                                             14          0.1510         0.0980                                             17          0.2460         0.1310                                             21          0.3980         0.2110                                             24          0.7090         0.3460                                             27          1.0660         0.5010                                             ______________________________________                                    

EXAMPLE 2

Liposomes containing entrapped oligonucleotide ON3 are prepared usingthe procedure described in Example 1, but replacing the lipid mixtureused in that Example by hydrogenated phosphatidylinositol, distearoylphosphatidylcholine and cholesterol in a molar ratio of 1:10:5.

The ability of the liposomes to inhibit uptake of oligonucleotide ON3 bymurine macrophage-like J774 cells is tested using the procedure of Nambaet al, 1992, Life Sciences, 50, 1773-1779, with minor modifications.After an incubation period of 360 minutes, the uptake of ON3 by the J774cells is 0.583%. When the procedure is repeated, replacing the liposomesby a solution of ON3 in phosphate buffered saline, the uptake of ON3 bythe J774 cells is 8.714%.

EXAMPLE 3

Liposomes containing entrapped oligonucleotide ON3 are prepared usingthe procedure described in Example 1, but replacing the lipid mixtureused in that Example by ganglioside GM₁ (ex Sigma Chemicals), distearoylphosphatidylcholine and cholesterol in a molar ratio of 1:10:5 and usinga 2:1 (by volume) mixture of methanol:chloroform, instead of chloroformalone, as the solvent for the lipids. The liposomes are tested as inExample 2: after an incubation period of 360 minutes, the uptake of ON3by J774 cells is 0.422%.

    __________________________________________________________________________    #             SEQUENCE LISTING                                                - <160> NUMBER OF SEQ ID NOS: 25                                              - <210> SEQ ID NO 1                                                           <211> LENGTH: 20                                                              <212> TYPE: DNA                                                               <213> ORGANISM: Artificial Sequence                                           <220> FEATURE:                                                                #Sequence:R INFORMATION: Description of Artificial                                  oligonucleotide                                                         <220> FEATURE:                                                                <223> OTHER INFORMATION: phosphorothioate backbones                           <220> FEATURE:                                                                #prepared withFORMATION: alternative oligonucleotide                                methoxy group substituting 2' sug - #ar moiety                          - <400> SEQUENCE: 1                                                           # 20               ttaa                                                       - <210> SEQ ID NO 2                                                           <211> LENGTH: 20                                                              <212> TYPE: DNA                                                               <213> ORGANISM: Artificial Sequence                                           <220> FEATURE:                                                                #Sequence:R INFORMATION: Description of Artificial                                  oligonucleotide                                                         <220> FEATURE:                                                                <223> OTHER INFORMATION: phosphorothioate backbone                            <220> FEATURE:                                                                #having methoxyORMATION: alternative oligonucleotide                          #at 2' positionsituting for sugar moiety                                      - <400> SEQUENCE: 2                                                           # 20               ttct                                                       - <210> SEQ ID NO 3                                                           <211> LENGTH: 20                                                              <212> TYPE: DNA                                                               <213> ORGANISM: Artificial Sequence                                           <220> FEATURE:                                                                #Sequence:R INFORMATION: Description of Artificial                                  oligonucleotide                                                         <220> FEATURE:                                                                <223> OTHER INFORMATION: phosphorotioate backbone                             <220> FEATURE:                                                                #with uniformNFORMATION: alternative oligonucleotide                                phosphorothiate backbone and nucleotide - #s 1-7 and                    #at the 2'0 being substituted by methoxy                                            position of the sugar moiety                                            <220> FEATURE:                                                                #with uniformNFORMATION: alternative oligonucleotide                                phosphorothiate backbone and nucleotide - #s 1-6 and                    #at the 2'0 being substituted by methoxy                                            position of the sugar moiety                                            <220> FEATURE:                                                                #with uniformNFORMATION: alternative oligonucleotide                                phosphorthiate backbone and nucleotides - # 1-5 and                     #at the 2 ' being substituted by methoxy                                            position of the sugar moiety                                            <220> FEATURE:                                                                #with uniformNFORMATION: alternative oligonucleotide                                phosphorothioate backbones and nucleoti - #des 1-6 and                        15-20 being substituted at the 2'- # position of the                          sugar moiety by propoxy                                                 <220> FEATURE:                                                                #backbones withORMATION: alternative phosphorothioate                               nucleotides 1-6 and 15-20 substitute - #d at the 2'                     #fluoroosition of the sugar moiety by                                         <220> FEATURE:                                                                #prepared withFORMATION: alternative oligonucleotide                                nucleotides 1-6 and 15-20 have ph - #osphodiester                             backbone and 2'-propoxy substitution - #s                               <220> FEATURE:                                                                #with nucleotidesMATION: alternative oligonucleotide                          #backbones and15-20 having phosphodiester                                           2'-methoxyethoxy substitutents                                          - <400> SEQUENCE: 3                                                           # 20               catt                                                       - <210> SEQ ID NO 4                                                           <211> LENGTH: 20                                                              <212> TYPE: DNA                                                               <213> ORGANISM: Artificial Sequence                                           <220> FEATURE:                                                                #Sequence:R INFORMATION: Description of Artificial                                  oligonucleotide                                                         <220> FEATURE:                                                                <223> OTHER INFORMATION: phosphorothioate backbone                            - <400> SEQUENCE: 4                                                           # 20               ctct                                                       - <210> SEQ ID NO 5                                                           <211> LENGTH: 20                                                              <212> TYPE: DNA                                                               <213> ORGANISM: Artificial Sequence                                           <220> FEATURE:                                                                <223> OTHER INFORMATION: Description of Artificial                                  Sequence:oliigonucleotide                                               <220> FEATURE:                                                                <223> OTHER INFORMATION: phosphorothioate backbone                            <220> FEATURE:                                                                #havingTHER INFORMATION: alternative oligonucleotide                          #substituted by a propoxybone and uniformly                                   #the sugar moietye 2' position of                                             - <400> SEQUENCE: 5                                                           # 20               ggcg                                                       - <210> SEQ ID NO 6                                                           <211> LENGTH: 20                                                              <212> TYPE: DNA                                                               <213> ORGANISM: Artificial Sequence                                           <220> FEATURE:                                                                #Sequence:R INFORMATION: Description of Artificial                                  oligonucleotide                                                         <220> FEATURE:                                                                <223> OTHER INFORMATION: phosphorothioate backbone                            <220> FEATURE:                                                                #with uniformNFORMATION: alternative oligonucleotide                                phosphorothiate backbones and nucleotid - #es 1-11                            being substituted by methoxy at t - #he 2' position of                        the sugar moiety                                                        - <400> SEQUENCE: 6                                                           # 20               gggt                                                       - <210> SEQ ID NO 7                                                           <211> LENGTH: 20                                                              <212> TYPE: DNA                                                               <213> ORGANISM: Artificial Sequence                                           <220> FEATURE:                                                                #Sequence:R INFORMATION: Description of Artificial                                  oligonucleotide                                                         <220> FEATURE:                                                                <223> OTHER INFORMATION: phosphorothioate backbone                            <220> FEATURE:                                                                #with uniformNFORMATION: alternative oligonucleotide                                phosphorothiate backbone with nucleotid - #es 10-20                           being substituted by methoxy at t - #he 2' position of                        the sugar moiety                                                        - <400> SEQUENCE: 7                                                           # 20               cccc                                                       - <210> SEQ ID NO 8                                                           <211> LENGTH: 20                                                              <212> TYPE: DNA                                                               <213> ORGANISM: Artificial Sequence                                           <220> FEATURE:                                                                #Sequence:R INFORMATION: Description of Artificial                                  oligonucleotide                                                         <220> FEATURE:                                                                #with uniformNFORMATION: alternative oligonucleotide                                phosphorothiotate backbones and nucleot - #ides  7-20                         being substituted by methoxy at t - #he 2' position of                        the sugar moiety                                                        - <400> SEQUENCE: 8                                                           # 20               ccct                                                       - <210> SEQ ID NO 9                                                           <211> LENGTH: 20                                                              <212> TYPE: DNA                                                               <213> ORGANISM: Artificial Sequence                                           <220> FEATURE:                                                                #Sequence:R INFORMATION: Description of Artificial                                  oligonucleotide                                                         <220> FEATURE:                                                                <223> OTHER INFORMATION: phosphorothioate backbone                            <220> FEATURE:                                                                #substituted at theTION: alternative oligonucleotide                                2' position of the sugar moiet - #y by a methoxy group                  <220> FEATURE:                                                                #prepared with aRMATION: alternative oligonucleotide                                phosphorothioate backbone and is uni - #formly                                substituted by fluoro at the 2'- # position of the                            sugar moiety                                                            - <400> SEQUENCE: 9                                                           # 20               tcgg                                                       - <210> SEQ ID NO 10                                                          <211> LENGTH: 20                                                              <212> TYPE: DNA                                                               <213> ORGANISM: Artificial Sequence                                           <220> FEATURE:                                                                #Sequence:R INFORMATION: Description of Artificial                                  oligonucleotide                                                         <220> FEATURE:                                                                <223> OTHER INFORMATION: phosphorothioate backbone                            <220> FEATURE:                                                                #includes uniformMATION: alternative oligonucleotide                                phosphorothiate backbone with nucleotid - #es 8-15                            substituted by methoxy at the 2'- # position of the                           sugar moiety                                                            <220> FEATURE:                                                                #prepared withFORMATION: alternative oligonucleotide                                uniform phosphorothioate backbones with - # nucleotides 8-20                  being substituted at the 2' po - #sition of the sugar                         moiety by flouro                                                        <220> FEATURE:                                                                #having nucleotidesTION: alternative oligonucleotide                                8-20 phosphodiester backbones and 2'- #-propoxy                               substitutions                                                           - <400> SEQUENCE: 10                                                          # 20               tctc                                                       - <210> SEQ ID NO 11                                                          <211> LENGTH: 20                                                              <212> TYPE: DNA                                                               <213> ORGANISM: Artificial Sequence                                           <220> FEATURE:                                                                #Sequence:R INFORMATION: Description of Artificial                                  oligonucleotide                                                         <220> FEATURE:                                                                <223> OTHER INFORMATION: oligonucleotide has phosphor -                       #othiate backbones,                                                           #15-20 areeotides at positions 1-5 and                                              substituted by methoxy at the 2'- # position of the                           sugar moiety                                                            - <400> SEQUENCE: 11                                                          # 20               ctcc                                                       - <210> SEQ ID NO 12                                                          <211> LENGTH: 20                                                              <212> TYPE: DNA                                                               <213> ORGANISM: Artificial Sequence                                           <220> FEATURE:                                                                #Sequence:R INFORMATION: Description of Artificial                                  oligonucleotide                                                         <220> FEATURE:                                                                #phosphorothiateRMATION: oligonucleotide has uniform                                backbones, nucleotides 1-12 are subs - #tituted by                      #the sugar moietythe 2' position of                                           - <400> SEQUENCE: 12                                                          # 20               gcag                                                       - <210> SEQ ID NO 13                                                          <211> LENGTH: 20                                                              <212> TYPE: DNA                                                               <213> ORGANISM: Artificial Sequence                                           <220> FEATURE:                                                                #Sequence:R INFORMATION: Description of Artificial                                  oligonucleotide                                                         <220> FEATURE:                                                                #phosphorothiateRMATION: oligonucleotide has uniform                          #substituted byand nucleotides 10-20 are                                      #the sugar moietythe 2' position of                                           - <400> SEQUENCE: 13                                                          # 20               cctc                                                       - <210> SEQ ID NO 14                                                          <211> LENGTH: 20                                                              <212> TYPE: DNA                                                               <213> ORGANISM: Artificial Sequence                                           <220> FEATURE:                                                                #Sequence:R INFORMATION: Description of Artificial                                  oligonucleotide                                                         - <400> SEQUENCE: 14                                                          # 20               gtgg                                                       - <210> SEQ ID NO 15                                                          <211> LENGTH: 20                                                              <212> TYPE: DNA                                                               <213> ORGANISM: Artificial Sequence                                           <220> FEATURE:                                                                #Sequence:R INFORMATION: Description of Artificial                                  oligonucleotide                                                         - <400> SEQUENCE: 15                                                          # 20               ccac                                                       - <210> SEQ ID NO 16                                                          <211> LENGTH: 20                                                              <212> TYPE: DNA                                                               <213> ORGANISM: Artificial Sequence                                           <220> FEATURE:                                                                #Sequence:R INFORMATION: Description of Artificial                                  oligonucleotide                                                         - <400> SEQUENCE: 16                                                          # 20               ccag                                                       - <210> SEQ ID NO 17                                                          <211> LENGTH: 20                                                              <212> TYPE: DNA                                                               <213> ORGANISM: Artificial Sequence                                           <220> FEATURE:                                                                #Sequence:R INFORMATION: Description of Artificial                                  oligonucleotide                                                         - <400> SEQUENCE: 17                                                          # 20               gtag                                                       - <210> SEQ ID NO 18                                                          <211> LENGTH: 20                                                              <212> TYPE: DNA                                                               <213> ORGANISM: Artificial Sequence                                           <220> FEATURE:                                                                #Sequence:R INFORMATION: Description of Artificial                                  oligonucleotide                                                         - <400> SEQUENCE: 18                                                          # 20               ttca                                                       - <210> SEQ ID NO 19                                                          <211> LENGTH: 20                                                              <212> TYPE: DNA                                                               <213> ORGANISM: Artificial Sequence                                           <220> FEATURE:                                                                #Sequence:R INFORMATION: Description of Artificial                                  oligonucleotide                                                         - <400> SEQUENCE: 19                                                          # 20               gcca                                                       - <210> SEQ ID NO 20                                                          <211> LENGTH: 20                                                              <212> TYPE: DNA                                                               <213> ORGANISM: Artificial Sequence                                           <220> FEATURE:                                                                #Sequence:R INFORMATION: Description of Artificial                                  oligonucleotide                                                         - <400> SEQUENCE: 20                                                          # 20               caca                                                       - <210> SEQ ID NO 21                                                          <211> LENGTH: 20                                                              <212> TYPE: DNA                                                               <213> ORGANISM: Artificial Sequence                                           <220> FEATURE:                                                                #Sequence:R INFORMATION: Description of Artificial                                  oligonucleotide                                                         - <400> SEQUENCE: 21                                                          # 20               ccgg                                                       - <210> SEQ ID NO 22                                                          <211> LENGTH: 20                                                              <212> TYPE: DNA                                                               <213> ORGANISM: Artificial Sequence                                           <220> FEATURE:                                                                <223> OTHER INFORMATION: Description of Artificial                                  Sequence:oligionucleotide                                               - <400> SEQUENCE: 22                                                          # 20               tggg                                                       - <210> SEQ ID NO 23                                                          <211> LENGTH: 20                                                              <212> TYPE: DNA                                                               <213> ORGANISM: Artificial Sequence                                           <220> FEATURE:                                                                #Sequence:R INFORMATION: Description of Artificial                                  oligonucleotide                                                         - <400> SEQUENCE: 23                                                          # 20               gctg                                                       - <210> SEQ ID NO 24                                                          <211> LENGTH: 20                                                              <212> TYPE: DNA                                                               <213> ORGANISM: Artificial Sequence                                           <220> FEATURE:                                                                #Sequence:R INFORMATION: Description of Artificial                                  oligonucleotide                                                         - <400> SEQUENCE: 24                                                          # 20               tggt                                                       - <210> SEQ ID NO 25                                                          <211> LENGTH: 20                                                              <212> TYPE: DNA                                                               <213> ORGANISM: Artificial Sequence                                           <220> FEATURE:                                                                #Sequence:R INFORMATION: Description of Artificial                                  oligonucleotide                                                         - <400> SEQUENCE: 25                                                          # 20               gagg                                                       __________________________________________________________________________

What is claimed is:
 1. A pharmaceutical composition comprising (A) anoligonucleotide 8 to 50 nucleotides in length, which is targeted to mRNAencoding human raf and inhibits raf expression, entrapped in (B)sterically stabilised liposomes.
 2. The composition according to claim1, in which at least one nucleotide of the oligonucleotide (A) ismodified at the 2' position of the sugar moiety.
 3. The compositionaccording to claim 1, in which the oligonucleotide (A) is a chimericoligonucleotide which contains a first region having at least onenucleotide modified to enhance target affinity and a second region whichis a substrate for RNAse H.
 4. The composition according to claim 3, inwhich a nucleotide modified to enhance target affinity is modified atthe 2' position of the sugar moiety.
 5. The composition according toclaim 2, in which the modified nucleotide has an alkoxy, alkoxyalkoxy orfluoro substituent at the 2' position.
 6. The composition according toclaim 3, in which the oligonucleotide (A) is a chimeric oligonucleotideand the region which is a substrate for RNAse H comprises at least one2'-deoxynucleotide.
 7. The composition according to claim 1, in whichthe oligonucleotide (A) has at least one phosphorothioate linkage. 8.The composition according to claim 1, in which, in the oligonucleotide(A), all nucleotides are 2'-deoxynucleotides and all backbone linkagesare phosphorothioate linkages.
 9. The composition according to claim 1,in which the oligonucleotide (A) is a chimeric oligonucleotide havingone or more regions with 2'-deoxynucleotides and one or more regionswith 2'-alkoxyalkoxynucleotides.
 10. The composition according to claim9, in which the 2'-alkoxyalkoxynucleotides are2'-methoxyethoxynucleotides.
 11. The composition according to claim 9,in which the one or more regions with 2'-deoxynucleotides havephosphorothioate backbone linkages and the one or more regions with2'-alkoxyalkoxynucleotides have phosphodiester backbone linkages. 12.The composition according to claim 9, in which the oligonucleotide (A)comprises a region of 2'-deoxynucleotides between two regions of2'-alkoxyalkoxynucleotides.
 13. The composition according to claim 1,wherein the oligonucleotide (A) is targeted to mRNA encoding humanA-raf.
 14. The composition according to claim 1, in which theoligonucleotide (A) is targeted to mRNA encoding human c-raf.
 15. Thecomposition according to claim 14, in which the oligonucleotide (A) istargeted to a translation initiation site, 3' untranslated region or 5'untranslated region of mRNA encoding human c-raf.
 16. The compositionaccording to claim 1 in which the oligonucleotide (A) comprises anucleotide sequence

    GCTCCATTGATGCAGCTTAA                                                                              (SEQ ID NO:1)                                             or                                                                            GATGCAGCTTAAACAATTCT                                                                                 (SEQ ID NO:2)                                          or                                                                            TCCCGCCTGTGACATGCATT                                                                                 (SEQ ID NO:3)                                          or                                                                            GTCTGGCGCTGCACCACTCT                                                                                 (SEQ ID NO:4)                                          or                                                                            CGCTCCTCCTCCCCGCGGCG                                                                                 (SEQ ID NO:5)                                          or                                                                            TCCTCCTCCCCGCGGCGGGT                                                                                 (SEQ ID NO:6)                                          or                                                                            CTCGCCCGCTCCTCCTCCCC                                                                                 (SEQ ID NO:7)                                          or                                                                            CTGGCTTCTCCTCCTCCCCT                                                                                 (SEQ ID NO:8)                                          or                                                                            CGGGAGGCGGTCACATTCGG                                                                                 (SEQ ID NO:9)                                          or                                                                            TCTGGCGCTGCACCACTCTC                                                                                 (SEQ ID NO:10)                                         or                                                                            TTCTCGCCCGCTCCTCCTCC                                                                                 (SEQ ID NO:11)                                         or                                                                            TTCTCCTCCTCCCCTGGCAG                                                                                 (SEQ ID NO:12)                                         or                                                                            CCTGCTGGCTTCTCCTCCTC                                                                                 (SEQ ID NO:13).                                    


17. The composition according to claim 1, in which the liposomes (B)comprise at least one underivatised vesicle-forming lipid and at leastone vesicle-forming lipid which is derivatised with a hydrophilicpolymer.
 18. The composition according to claim 17, in which thehydrophilic polymer is a polyethyleneglycol.
 19. The compositionaccording to claim 17, in which the derivatised lipid is a phospholipidhaving an amino group.
 20. The composition according to claim 19, inwhich the hydrophilic polymer is attached to the phospholipid through acarbamate linkage.
 21. The composition according to claim 19, in whichthe amino-containing phospholipid is a phosphatidylethanolamine.
 22. Thecomposition according to claim 21, in which the amino-containingphospholipid is distearoyl phosphatidylethanolamine.
 23. The compositionaccording to claim 17, in which the derivatised lipid comprises 1-20mole % of the total lipid content of the liposomes.
 24. The compositionaccording to claim 17, in which the underivatised lipid is a lipidhaving two hydrocarbon chains and a polar head group and/or a sterol.25. The composition according to claim 24, in which the lipid having twohydrocarbon chains and a polar head group is a phosphatidylcholine. 26.The composition according to claim 25, in which the phosphatidylcholineis distearoyl phosphatidylcholine.
 27. The composition according toclaim 24, in which the sterol is cholesterol.
 28. The compositionaccording to claim 17, in which the liposomes comprise 4-10 mol %derivatised lipid, 40-80 mol % underivatised lipid and 20-50 mol %sterol.
 29. The composition according to claim 28, in which the molarratio of derivatised lipid: underivatised lipid: sterol is 1:10:5. 30.The composition according to claim 1, in which the liposomes (B)comprise (i) a glycolipid together with (ii) a vesicle-formingphospholipid or sphingolipid or mixture thereof and, optionally, (iii) asterol and/or an acylglycerol lipid.
 31. The composition according toclaim 30, in which the glycolipid is a negatively charged glycolipid.32. The composition according to claim 31, in which the liposomescomprise (i) a negatively charged glycolipid together with (ii) avesicle-forming phospholipid and/or sphingolipid and (iii) a sterol oracylglycerol lipid.
 33. The composition according to claim 31, in whichthe glycolipid is ganglioside GM₁ or hydrogenated phosphatidylinositol.34. The composition according to claim 30, in which the vesicle-formingphospholipid is a phosphatidylcholine or a phosphatidylethanolamine. 35.The composition according to claim 34, in which the phospholipid isdistearoyl phosphatidylcholine or dioleoyl phosphatidylethanolamine. 36.The composition according to claim 30, in which the sphingolipid issphingomyelin.
 37. The composition according to claim 30, in which theacylglycerol lipid has two fatty acid acyl groups each having at least12 carbon atoms and one acyl group of formula R¹ CO--, where R¹ is aresidue, containing up to 10 carbon atoms, of a monocarboxylic acid offormula R¹ COOH after removal of the --COOH group, or of formula OC--R²--COOH where R² is a residue, containing up to 10 carbon atoms, of adicarboxylic acid of formula HOOC--R² --COOH after removal of both--COOH groups.
 38. The composition according to claim 37, in which theacylglycerol is 1,2-dipalmitoyl-sn-3-succinyl glycerol.
 39. Thecomposition according to claim 30, in which the liposomes comprise 2 to20 mol % of the glycolipid, 40 to 80 mol % of the phospholipid,sphingolipid or mixture thereof and 10 to 50 mol % of the sterol or 10to 30 mol % of the acylglycerol.
 40. The composition according to claim39, in which the liposomes comprise 4 to 10 mol % of the glycolipid, 60to 80 mol % of the phospholipid, sphingolipid or mixture thereof and 20to 40 mol % of the sterol or 10 to 30 mol % of the acylglycerol.
 41. Thecomposition according to claim 1, in which the liposomes have an averageparticle size of 50 to 200 nm.
 42. The composition according to claim41, in which the liposomes have an average particle size of 80 to 120nm.
 43. A method of inhibiting the expression of human raf whichcomprises contacting tissues or cells which express human raf with acomposition according to claim
 1. 44. A method of treating mammaliancancer which comprises administering a composition according to claim 1to a mammal in need of such treatment.